ISG15 Promotes ERK1 ISGylation, CD8+ T Cell Activation and Suppresses Ovarian Cancer Progression.

Increased number of tumor-infiltrating CD8+ lymphocytes is associated with improved survival in patients with advanced stage high grade serous ovarian cancer (HGSOC) but the underlying molecular mechanism has not been thoroughly explored. Using transcriptome profiling of microdissected HGSOC tissue with high and low CD8+ lymphocyte count and subsequent validation studies, we demonstrated that significantly increased ISG15 (Interferon-stimulated gene 15) expression in HGSOC was associated with high CD8+ lymphocyte count and with the improvement in median overall survival in both univariate and multivariate analyses. Further functional studies showed that endogenous and exogenous ISG15 suppressed ovarian cancer progression through ISGylation of ERK in HGSOC, and activation of NK cells and CD8+ T lymphocytes. These data suggest that the development of treatment strategies based on up-regulating ISG15 in ovarian cancer cells or increased circulating ISG15 in ovarian cancer patients is warranted.

Human omental-derived adipose stem cells increase ovarian cancer proliferation, migration, and chemoresistance.

OBJECTIVES: Adipose tissue contains a population of multipotent adipose stem cells (ASCs) that form tumor stroma and can promote tumor progression. Given the high rate of ovarian cancer metastasis to the omental adipose, we hypothesized that omental-derived ASC may contribute to ovarian cancer growth and dissemination. MATERIALS AND METHODS: We isolated ASCs from the omentum of three patients with ovarian cancer, with (O-ASC4, O-ASC5) and without (O-ASC1) omental metastasis. BM-MSCs, SQ-ASCs, O-ASCs were characterized with gene expression arrays and metabolic analysis. Stromal cells effects on ovarian cancer cells proliferation, chemoresistance and radiation resistance was evaluated using co-culture assays with luciferase-labeled human ovarian cancer cell lines. Transwell migration assays were performed with conditioned media from O-ASCs and control cell lines. SKOV3 cells were intraperitionally injected with or without O-ASC1 to track in-vivo engraftment. RESULTS: O-ASCs significantly promoted in vitro proliferation, migration chemotherapy and radiation response of ovarian cancer cell lines. O-ASC4 had more marked effects on migration and chemotherapy response on OVCA 429 and OVCA 433 cells than O-ASC1. Analysis of microarray data revealed that O-ASC4 and O-ASC5 have similar gene expression profiles, in contrast to O-ASC1, which was more similar to BM-MSCs and subcutaneous ASCs in hierarchical clustering. Human O-ASCs were detected in the stroma of human ovarian cancer murine xenografts but not uninvolved ovaries. CONCLUSIONS: ASCs derived from the human omentum can promote ovarian cancer proliferation, migration, chemoresistance and radiation resistance in-vitro. Furthermore, clinical O-ASCs isolates demonstrate heterogenous effects on ovarian cancer in-vitro.

TGF-ß modulates ovarian cancer invasion by upregulating CAF-derived versican in the tumor microenvironment.

TGF-ß has limited effects on ovarian cancer cells, but its contributions to ovarian tumor growth might be mediated through elements of the tumor microenvironment. In the present study, we tested the hypothesis that TGF modulates ovarian cancer progression by modulating the contribution of cancer-associated fibroblasts (CAF) that are present in the microenvironment. Transcriptome profiling of microdissected stromal and epithelial components of high-grade serous ovarian tumors and TGF-ß-treated normal ovarian fibroblasts identified versican (VCAN) as a key upregulated target gene in CAFs. Functional evaluations in coculture experiments showed that TGF-ß enhanced the aggressiveness of ovarian cancer cells by upregulating VCAN in CAFs. VCAN expression was regulated in CAFs through TGF-ß receptor type II and SMAD signaling. Upregulated VCAN promoted the motility and invasion of ovarian cancer cells by activating the NF-κB signaling pathway and by upregulating expression of CD44, matrix metalloproteinase-9, and the hyaluronan-mediated motility receptor. Our work identified a TGF-ß-inducible gene signature specific to CAFs in advanced high-grade serous ovarian tumors, and showed how TGF-ß stimulates ovarian cancer cell motility and invasion by upregulating the CAF-specific gene VCAN. These findings suggest insights to develop or refine strategies for TGF-ß-targeted therapy of ovarian cancer.

Identification of FGFR4 as a potential therapeutic target for advanced-stage, high-grade serous ovarian cancer.

PURPOSE: To evaluate the prognostic value of fibroblast growth factor receptor 4 (FGFR4) protein expression in patients with advanced-stage, high-grade serous ovarian cancer, delineate the functional role of FGFR4 in ovarian cancer progression, and evaluate the feasibility of targeting FGFR4 in serous ovarian cancer treatment. EXPERIMENTAL DESIGN: Immunolocalization of FGFR4 was conducted on 183 ovarian tumor samples. The collected FGFR4 expression data were correlated with overall survival using Kaplan-Meier and Cox regression analyses. The effects of FGFR4 silencing on ovarian cancer cell growth, survival, invasiveness, apoptosis, and FGF1-mediated signaling pathway activation were evaluated by transfecting cells with FGFR4-specific siRNAs. An orthotopic mouse model was used to evaluate the effect of injection of FGFR4-specific siRNAs and FGFR4 trap protein encapsulated in nanoliposomes on ovarian tumor growth in vivo. RESULTS: Overexpression of FGFR4 protein was significantly associated with decreased overall survival durations. FGFR4 silencing significantly decreased the proliferation, survival, and invasiveness and increased apoptosis of ovarian cancer cells. Also, downregulation of FGFR4 significantly abrogated the mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), and WNT signaling pathways, which are activated by FGF1. Targeting FGFR4 with the FGFR4-specific siRNAs and FGFR4 trap protein significantly decreased ovarian tumor growth in vivo. CONCLUSIONS: FGFR4 is a prognostic marker for advanced-stage, high-grade serous ovarian carcinoma. Silencing FGFR4 and inhibiting ligand-receptor binding significantly decrease ovarian tumor growth both in vitro and in vivo, suggesting that targeting ovarian cancer cells with high levels of FGFR4 protein expression is a new therapeutic modality for this disease and will improve survival of it.

Chapter 18: Sensing cytoskeletal mechanics by ballistic intracellular nanorheology (BIN) coupled with cell transfection.

Key processes in normal and diseased cells depend directly or indirectly on the viscoelastic properties of the cytoplasm. Particle-tracking microrheology is a highly versatile method that measures the viscoelastic properties of cytoplasm directly by tracking fluorescent nanoparticles embedded in the cytoskeleton with high spatial and temporal resolutions. Here we present a new method that combines cell transfection, ballistic injection, and particle-tracking microrheology to monitor changes in cytoplasmic micromechanics following controlled changes in protein expression. We demonstrate that cells transfected with GFP (green fluorescent protein) display viscoelastic properties identical to untransfected fibroblasts, that low levels of expression of GFP-alpha-actinin do not affect cell microrheology, and that the transient transfection with GFP-C3 transferase reduces the elasticity of the cytoplasm of fibroblasts to a similar extent as C3 transferase toxin, which de-activates the GTPase Rho. Combining cell transfection with particle-tracking microrheology opens the way to quantitative, single live-cell mechanical studies where stable cell lines cannot be easily established, but where commonly used transfections can be exploited to manipulate cytoskeletal organization.

Single-molecule analysis of cadherin-mediated cell-cell adhesion.

Cadherins are ubiquitous cell surface molecules that are expressed in virtually all solid tissues and localize at sites of cell-cell contact. Cadherins form a large and diverse family of adhesion molecules, which play a crucial role in a multitude of cellular processes, including cell-cell adhesion, motility, and cell sorting in maturing organs and tissues, presumably because of their different binding capacity and specificity. Here, we develop a method that probes the biochemical and biophysical properties of the binding interactions between cadherins expressed on the surface of living cells, at the single-molecule level. Single-molecule force spectroscopy reveals that classical cadherins, N-cadherin and E-cadherin, form bonds that display adhesion specificity, and a pronounced difference in adhesion force and reactive compliance, but not in bond lifetime. Moreover, their potentials of interaction, derived from force-spectroscopy measurements, are qualitatively different when comparing the single-barrier energy potential for the dissociation of an N-cadherin-N-cadherin bond with the double-barrier energy potential for an E-cadherin-E-cadherin bond. Together these results suggest that N-cadherin and E-cadherin molecules form homophilic bonds between juxtaposed cells that have significantly different kinetic and micromechanical properties.